Spot test (lichen)
A spot test in lichenology is a spot analysis used to help identify lichens.: 369 It is performed by placing a drop of a chemical on different parts of the lichen and noting the color change (or lack thereof) associated with application of the chemical. The tests are routinely encountered in dichotomous keys for lichen species, and they take advantage of the wide array of secondary metabolites produced by lichens and their uniqueness among taxa. As such, spot tests reveal the presence or absence of chemicals in various parts of a lichen. They were first proposed by the botanist William Nylander in 1866.
The three most common spot tests are:
- K test: a 10% aqueous solution of potassium hydroxide (KOH), or, in the absence of KOH, a 10% aqueous solution of sodium hydroxide (NaOH, lye), which provides nearly identical results;
- C test: A 5.25% solution of sodium hypochlorite or undiluted household bleach; and
- P test: an ethanolic solution of para-phenylenediamine, made by placing a drop of ethanol (70-95%) over a few crystals of the chemical (yielding an unstable solution).
Although the above tests are most common, a KC test may be performed by wetting the thallus with K followed immediately by C. In addition, Lugol’s iodine may be useful in identifying certain species.
Performing spot tests
Spot tests are performed by placing a small amount of the desired reagent on the portion of the lichen to be tested. Often, both the cortex and medulla of the lichen are tested, and at times it is useful to test other structures such as soralia. One method is to draw up a small amount of the chemical into a glass capillary and touch it to the lichen thallus. A razor blade may be used to remove the cortex and access the medulla.
Spot tests may be used individually or in combination. The results of a spot tests are typically represented with a short code that includes, in order, (1) a letter indicating the reagent used, (2) a "+" or "-" sign indicating a color change or lack of color change, respectively, and (3) a letter or word indicating the color observed. In addition, care should be taken to indicate which part of the lichen was tested. For example, "Cortex K+ orange, C-, P-" means the cortex of the test specimen turned orange with application of KOH and did not change under bleach or para-phenylenediamine. Similarly, "Medulla K-, KC+R" would indicate the medulla of the lichen was insensitive to application of KOH, but application of KOH followed immediately by bleach caused the medulla to turn red.
It may sometimes be useful to perform other diagnostic measures in addition to spot tests. For example, some lichen metabolites fluoresce under ultraviolet radiation such that exposing certain parts of the lichen to a UV light source can reveal the presence of absence of those metabolites similarly to spot tests. More advanced analytical techniques, such as thin layer chromatography, high performance liquid chromatography, and mass spectrometry may also be useful in initially characterizing the chemical composition of lichens or when spot tests are unrevealing.
Finnish lichenologist William Nylander is generally considered to have been the first to demonstrate the use of chemicals to help with lichen identification. In papers published in 1866, he suggested spot tests using KOH and bleaching powder to get characteristic colour reactions–typically yellow, red, or green. In the mid-1930s, Yasuhiko Asahina created the test with para-phenylendiamine, which gives yellow to red reactions with secondary metabolites that have a free aldehyde group.
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